Many molecular biology techniques depend on cloning individual cells from a mixture of cells.
For example, in the production of monoclonal antibodies, an essential step is hybridoma selection, including the separation and culture of individual hybridoma clones (fused myelomas and primary mouse cells). After cell fusion, the traditional way of selecting for monoclonality is to plate out single cells into 96-well dishes. This is repeated until clonality is assured.
Similarly, understanding gene function and identification of pharmaceutical leads requires the establishment of cell lines containing transfected genes expressed at an appropriate level. Standard techniques require the co-transfection of a gene with a dominant selectable marker followed by selection for growth for example in an antibiotic such as G418 or hygromycin. The resulting colonies are then picked by hand and further analysed for gene expression (RT-PCR) and functional expression.
Ascertaining optimal conditions for cell growth and differentiation requires broad testing of growth factors and culture conditions. The evaluation of a particular treatment requires a statistical approach on a large number of individual cells. One way to achieve this is to use numerous culture dishes, several for each treatment.
This process of cloning out may be modified and automated through the use of robots. Thus, for example, the ClonePix robot (manufactured by Genetix) implements this process by picking individual colonies directly from standard semi-solid media, the media preventing migration of the dividing cells. Thus, an imaging head captures images of colonies growing in the medium under white light, and software routines allow the separation and detection of individual colonies. A picking head then picks individual colonies into a 96-well plate.
Using a robot implemented picking method, colonies can be picked into 96-well plates at a picking speed of up to 400 clones per hour and graphic software allows the user to select colonies on the basis of size, shape, brightness and proximity. Furthermore, the software allows stratification of clones into slow, medium and fast growing cells, and clones of the same class may be grouped in the same 96-well plate. This gives rise to considerable savings in subsequent tissue culture steps as all wells can be processed at the same time.
However, this robot implemented cloning method relies on visualisation solely of colony size. Thus, the image capture only provides information on the size of the colony, and all colonies within a certain size range are picked. It is known for example that different hybridoma clones are capable of producing varying amounts of antibody. No information is provided or processed as to the productivity of different cells (i.e., the quantity of product produced or secreted), and this robot implemented cloning method therefore cannot discriminate between a high-producing hybridoma cell or colony and a low-producing hybridoma cell or colony. With regard to transfected cells, the robot cannot distinguish between clones with different levels of expression and/or secretion of recombinant protein.
A method disclosed in EP1752771 addresses this issue by identifying cells producing a polypeptide of interest using a combination of a class marker and a specificity marker. Marker-polypeptide complexes can then be detected, for example by an automated imaging system, and cells producing a high level of the polypeptide picked directly by a robot. Application of the class marker limits the diffusion of the secreted polypeptide and causes the formation of a halo or aura of polypeptide surrounding the cell or cell colony. Formation of such a halo increases the effective concentration of the polypeptide in the vicinity of the cell or colony, to enable more efficient binding by the specificity marker to the polypeptide. This method may allow the application of a smaller amount or concentration of specificity marker compared to methods which do not make use of a class marker.
However, there is a still a need for improved methods for selecting cells or colonies which produce a polypeptide of interest.